Method for determining microbial contamination

ABSTRACT

The present invention discloses a process for quantitative and/or qualitative determination of the microbial contamination of suspensions, emulsions or dispersions containing minerals and/or pigments and/or fillers and/or fiber materials wherein following the addition of one or more substances which can be degraded by microorganisms, mixing and optionally subsequent incubation a sample of the suspensions, emulsions or dispersions is centrifuged to separate the microorganisms from the minerals and/or fillers and/or pigments and/or fiber materials and the number and/or size and/or type of the microorganisms is determined in the aqueous supernatant phase after one or more incubations.

DESCRIPTION

1. Technical Field

The present invention relates to a process for quantitative and/orqualitative determination of the microbial contamination of aqueoussuspensions, emulsions or dispersions containing minerals and/orpigments and/or fillers and/or fiber materials, optionally incombination with polymers in colloidal form.

2. Related Art

Determinations of the germ contamination and hygienic controls ofaqueous suspensions by means of conventional methods essentially rely onthe propagative capacity of the microorganisms to be detected.Naturally, the time required to perform these methods ranges from 24 hto several days. These time periods are too long for many questions, andthe results are obtained too late to intervene in a guiding manner inproduction processes. Particularly performing controls prior totransport time and again requires methods which are characterized byshort determination intervals.

Thus, the main problem of conventional microbiological analyses ofaerobic mesophilic germs, such as Plate Count or Easicult, is their longincubation period of up to 48 h. By this methods it is impossible toobtain an evaluation and, thereby, determination of the germ countearlier than after the elapse of two days. In addition, several othereffects must be considered such as the nutrient medium, partial pressureof oxygen (aerobic/anaerobic), selectivity, pH and much more.

Therefore, it is often impossible to determine the germ count ofproducts, such as pigment slurries, prior to their shipment to customersand to intervene in a guiding manner by adding biocidal substances. Dueto the long transport times required of up to 6 weeks by deep sea shipor rail, the pigment slurry may deteriorate or become unusable. Thewhite pigment slurry may develop a gray color and start to smell. Todate, preventive over-dosage of biocidal substances in the pigmentslurry is the only possibility to exclude spoilage, is verycost-intensive, dissipates resources, and is ecologically nonsensical.Moreover, two different approaches are necessary to analyze for bothaerobic mesophilic germs and fungi. Furthermore, the preparation ofserial dilutions is necessary, thus multiplying the number of analyses.

Conventional methods for the determination of germs in the paper andpigment industries have been for example described in the“Schweizerisches Lebensmittelbuch”, chapter 56, section 7.01, 1985edition, 1988 revised version, “Bestimmung von aeroben Bakterien undKeimen”, and in the “Schweizerisches Lebensmittelbuch”, chapter 56,section 7.22, 1985 edition, 1988 revised version, “Bestimmung vonPilzen”. Generally, prior to performing a determination in each case anincubation period of about 48 hours is required.

The CellFacts® particle analyzer and method have been developed byMicrobial Systems company, Ltd. More detailed information may beobtained from Labor flash 9/96, Zeitung mit Leserdienst für Labor undForschung, Ott Verlag+Druck AG, Ch-3607 Thun, Switzerland. Thus, it hasbeen mentioned that the CellFacts analyzer may also be used to carry outdeterminations of the germ count in calcium carbonate slurries. However,experiments performed in the applicant's laboratory revealed that adetermination of this type in the manner described by the manufactureris impossible or only inaccurate.

The principle of the measurement performed by the CellFacts analyzer isbased on the measurement of bacteria, fungi, and yeasts in the form ofparticles in an electrical field wherein the number of particles isdetermined by an interval incubation of the samples and subsequentmeasurement of the increase in particle count and, in the case ofexponential growth, by extrapolation to t₀. This measurement principleis also effective in measuring a “low” number of “foreign particles”having the same or a similar size as bacteria, fungi, and yeasts. In thecase of suspensions and emulsions containing a proportion of “foreignparticles”, such as minerals, fillers and/or pigments, of >1% by wt. thenumber of inert “foreign particles” present having the same size asmicroorganisms, namely 0.5-20 μm, i.e. the blank value t₀, is too highto enable the detection of a further increase of the germs bypropagation in the incubator within a period of <10 hours. Thus, theCellFacts analyzer is unable to perform a sufficiently exactmeasurement, and the manufacturer requires a dilution of the startingliquid to ensure applicability.

At a blank value of 10⁸ particles/ml, it is impossible to significantlydetect an increase of 10³ particles/ml by means of the CellFactsanalyzer. However, the dilution required due to the presence of the“foreign particles” is too high so that the dilution of the reproductiveorganisms which of course are diluted by the same order of magnitude isno longer significant, and the result obtained is incorrect.Furthermore, “foreign particles” having a diameter of >20 μm may plugthe measurement cell which has a diameter of only 30 μm. The “foreignparticles” may for example be of mineral type, such as calciumcarbonate, synthetic, organic, of polymeric type, such as polystyreneacrylate dispersions, or of natural, organic type, such as starchsolutions or hemicelluloses and/or cellulose fibers, or a combination ofthe above particles as they are for example present in a paper millcycle.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a quick andpowerful, easily performed process for the quantitative and/orqualitative determination of the microbial contamination of aqueoussuspensions, emulsions or dispersions containing minerals and/orpigments and/or fillers and/or fiber materials wherein said processavoids the above described disadvantages of the prior art.

According to the present invention, this object has been solved by aprocess according to the generic part of claim 1 characterized in that asample of the suspensions, emulsions or dispersions, following theaddition of one or more organic substances which can be degraded bymicroorganisms and optionally following a subsequent incubation, iscentrifuged to separate the microorganisms from the minerals and/orfillers and/or pigments and/or fiber materials and the number and/orsize and/or nature of the microorganisms in the aqueous supernatantphase is determined after one or more incubations.

Preferred embodiments of the present invention will become obvious fromthe dependent Claims as well as the following Specification and theExamples.

DETAILED DESCRIPTION OF THE INVENTION

Since several years, the quantitative and qualitative determination ofmicroorganisms belongs to the prior art. However, the determination ofmicrobial contamination is particularly difficult in cases where a highconcentration of other solid particles such as minerals, pigments,fillers and/or fiber materials is present in the sample to beinvestigated. Because these “foreign particles” often have a sizesimilar to microorganisms, generally, it is only possible to minimizethe number of such “foreign particles” by carrying out high dilutions.However, concomitantly and unavoidably, these high dilution steps reducethe number of contaminating microorganisms, and a long incubation periodis required to increase the number of microorganisms by means ofpropagation to an extent that enables safe detection.

Therefore, there has been a need to create a new process of the typementioned in the beginning which provides reproducible and reliableresults with respect to the number, size and/or type of themicroorganisms in the sample to be investigated in a substantiallyshorter time.

Surprisingly and unexpectedly, it has been discovered that it ispossible to separate the microorganisms from inert materials (“foreignparticles”) by addition of degradable organic substances to the sampleand performing a subsequent centrifugation. Usually, the microorganismspreferably adhere to the surface of the mineral, pigment, filler and/orfiber materials making it difficult to separate them from the surface.Although the “foreign particles” are sedimented by a simplecentrifugation step, however, the microorganisms are drawn along intothe pellet because they preferably adhere to those particles, andthereby the portion of microorganisms remaining in the supernatantprovides incorrect values with respect to the degree of contaminationwith microorganisms.

It has been shown that the addition of biologically degradable organicsubstances surprisingly enables a separation of the microorganisms andthe minerals, pigments, fillers and/or fiber materials. Preferably, theorganic substance which can be degraded by microorganisms is a nutrientmedium conventionally used in the cultivation of microorganisms.Surprisingly, this nutrient medium acts as a separation agent betweenthe microorganisms and the foreign particles and, thus, for the firsttime enables the separation of and distinction between the two withoutaffecting further steps of the microorganism analysis. Substances whichmay not be biologically degraded or whose biological degradation isdifficult bear the risk that the microorganisms are separated from theinert particles while at the same time the substances act as inhibitorsof microorganism growth and thus lead to inaccurate results.

In the process according to the present invention not only one processstep, namely the dilution step, may be omitted but in addition theincubation period is extraordinarily reduced. Furthermore, there is notjust added a separation agent but the separation agent simultaneouslyacts as a nutrient solution which may be employed for optimalpropagation of the microorganisms to be investigated and, in a preferredembodiment, is selected to be specific for a particular microorganismtype to be tested, such as bacteria, fungi or yeasts.

It was only by the addition of an organic substance which may bedegraded by the microorganisms, preferably in the form of a nutrientsolution or medium, respectively, and by introduction of acentrifugation step that the object according to the present inventionhas been solved. A reduction in analysis time or even the possibility toperform an analysis at all has been achieved because the sample to betested contains a smaller number of “foreign particles”. Moreover, it ispossible to use a smaller starting number and a smaller increase inmicrobiological particles over time than with samples having a higherblank value, i.e. a higher number of “foreign particles”.

By the process according to the invention, aqueous suspensions,emulsions and dispersions may be investigated which among othersubstances contain minerals, pigments, fillers and/or fiber materials,such as cellulose fibers, and which optionally further contain polymerssuch as natural, synthetic or semisynthetic polymers in colloidal form.Examples of such polymers are: styrene butadiene, styrene acrylate,melamine resins, formaldehyde urea resins, starch,carboxymethylcellulose.

Preferably, the samples to be tested are derived from the paperprocessing industry. Further areas of use are the pigment industry andthe metal processing industry.

According to the present invention, a sample of the suspensions,emulsions or dispersions to be tested for microbial contamination istaken. Generally, the amount of the sample obtained is 0.5-20 ml,however, a smaller or higher amount may be taken. Besides, the amount ofsample taken is not important for the success of the process of theinvention.

The sample obtained is added and mixed with organic substances which maybe degraded by microorganisms. The amount of biodegradable organicsubstances added to the sample is 0.5-50 ml of biodegradable substanceper ml of sample.

The ratio of the sample volume to the volume of biodegradable substanceis dependent on the original concentration of the sample and theconcentration of biodegradable substance in solution. The ratio isadjusted to an amount of biodegradable substance which is large enoughto enable a separation of the microorganisms and the minerals, fillers,pigments and/or fiber materials and optionally also polymers incolloidal form. The optimal ratio of the sample volume to the volume ofbiodegradable substance which must be determined in each case may bedetermined by the skilled artisan by means of manual experimentation.

Usually the concentration of the solution containing the biodegradablesubstance is selected to achieve a ratio of the sample solution to thesolution containing the biodegradable substance of 1:0.1 to 1:100. Theoptimal ratio is strongly dependent on the concentration and thecomposition of the respective sample solution. Pigment and/or fillersuspensions having a solids content of >65% by wt. are generally dilutedin a ratio of 1:2 to 1:10, preferably in a ratio of 1:3. If the solidscontent is <65% by wt., the dilution is usually performed in a ratio of1:0.1 to 1:1. In certain cases, especially if a very high contaminationcan be expected, the dilution is generally performed in a ratio of 1:10to 1:100. The chosen dilution factor must be considered in thesubsequent evaluation of the contamination or must be pointed outspecifically in the result.

The biodegradable organic substances particularly include the group ofnutrient media which are specific for a particular species ofmicroorganism to be tested. Preferably, nutrient media contain a sourceof carbon, nitrogen, phosphate and/or sulfur as well as optionallyminerals, growth factors and/or vitamins. Other substances may be addedto the nutrient medium if they are required for optimal growth of themicroorganisms. In the following, there are described preferred mediawhich may be employed according to the present invention.

Optionally, following addition of the nutrient medium one or moreincubation steps may be carried out to enhance the number ofmicroorganisms in the sample. This may be especially advantageous for asubsequent qualitative analysis. In a preferred embodiment of theinvention, however, this incubation prior to centrifugation is omittedleading to a marked time reduction.

To separate the microorganisms from the minerals, fillers, pigmentsand/or fiber materials a centrifugation step is carried out followingthe addition of the nutrient medium and the optional subsequentincubation step. The centrifugation step is performed in a manner toremove most of the microorganisms, i.e. more than 50%, from the “foreignparticles”, i.e. the minerals, fillers, pigments, polymers, and fibermaterials. The centrifugation must be effective to accumulate themicroorganisms in the upper phase while the foreign particles aresedimented. For this purpose, the centrifugation is preferably carriedout at 100 to 1500 g, preferably 200 to 1200 g and particularlypreferred at 600 to 1000 g. The gravity field is adjusted depending onthe minerals, fillers, pigments, and fiber materials to be separated ordepending on the microorganisms to be separated, respectively.

The optimal sedimentation index may be determined by the skilled artisanby means of experimentation. The centrifugation itself is performed fora time of 1 to 30 minutes, preferably 2 to 15 minutes and particularlypreferred 5 to 10 minutes. The optimal centrifugation time may bedetermined by the skilled artisan performing laboratory experiments.

As the minerals and/or fillers and/or pigments there are preferablyused: compounds containing elements of the second and/or third maingroup and/or fourth main group and/or fourth side group of the periodicsystem of the elements, particularly calcium and/or silicon and/oraluminium and/or titanium and/or barium and/or organic pigments.

As the minerals, fillers, and pigments, there are preferably usedminerals and/or fillers and/or pigments containing kaolin and/oraluminium hydroxide and/or titanium hydroxide and/or barium sulfateand/or polystyrene hollow spheres and/or formaldehyde resins and/orcalcium carbonate, particularly natural calcium carbonates and/or marbleand/or lime and/or dolomite and/or calcium carbonates containingdolomite and/or synthetically prepared calcium carbonates, so-calledprecipitated calcium carbonated.

The supernatant containing the microorganisms is removed and may then beused directly in a quantitative and/or qualitative determination of themicrobial contamination. Preferably, this is followed by one or moreincubation steps to increase the number of microorganisms in thesupernatant. This propagation step is performed at an incubationtemperature which favors the microorganisms and is dependent on the typeof microorganisms to be propagated. Preferred incubation temperaturesare in the range of 20 to 37° C., further preferred 28 to 34° C., andparticularly preferred 31.5 to 32.5° C. The incubation temperatureswhich are necessary in each case are known to the skilled artisan andmay be either found in monographs or may be experimentally determined.

The total time of the individual incubation steps at an incubationtemperature of 30° C. is up to 12 hours at a contamination of less than10⁵ germs/ml of sample, up to 6 hours at a contamination of more than10⁵ germs/ml sample but less than 10⁶ germs/g of sample, and up to 3hours in a suspension with an original solids content of 60-80% by wt.and a contamination of more than 10⁵ germs/ml (using tryptic soy brothagar).

The sum of the individual incubation steps at 30° C. is up to 2 to 6 ata contamination of less than 10⁵ germs/ml of sample, 2 to 4 at acontamination of more than 10⁵ germs/ml sample but less than 10⁶ germs/gof sample, and 2 to 3 in a suspension with an original solids content of60-80% by wt. and a contamination of more than 10⁵ germs/ml (usingtryptic soy broth agar).

The individual incubation times at an incubation temperature of 30° C.are 1 to 12 hours at a contamination of less than 10⁵ germs/ml ofsample, 1 to 6 hours at a contamination of more than 10⁵ germs/ml samplebut less than 10⁶ germs/g of sample, and 1 to 2 hours in a suspensionwith an original solids content of 60-80% by wt. and a contamination ofmore than 10⁵ germs/ml (using tryptic soy broth agar).

Several methods known from the prior art are available for thequantitative or qualitative determination, respectively, of themicroorganisms thus obtained. Particularly preferred according to thepresent invention is the CellFacts® analysis or the ATP method. Othermethods are available and are known to those skilled in the art. Thepreferred methods are described in more detail in the followingExamples.

After the process according to the present invention has been carriedout, a qualitative and/or quantitative determination of themicroorganisms may be performed. First, qualitative determination meansa rough differentiation between the groups of fungi, yeasts andbacteria. If the CellFacts® analysis is calibrated which specificmicroorganisms such as specific bacteria also a further specificationwithin individual subtypes may be performed. The qualitative analysis,for example via the CellFacts® method, is performed on the basis of agross differentiation with respect to size or volume, respectively, of asingle cell.

The type of the nutrient media employed as the separation agentparticularly depends on the microorganism to be separated or propagated.Preferably, nutrient media are employed which enable the selectivegrowth of the germs to be determined and, if possible, suppress thegrowth of other germs not to be determined. Examples of nutrient mediawhich may be employed according to the present invention are tryptoneazolectin/Tween 20 (TAT) broth agar, glucose solution, peptone/caseinnutrient broth, preferably tryptic soy broth agar. The composition ofthe nutrient media is given in annex to this specification.

The concentration of the nutrient medium for the microorganisms ispreferably in the range of 0.1 to 10% by wt., more advantageously 2 to5% by wt., and particularly advantageous about 3% by wt.

The process according to the present invention not only enables aqualitative but also a quantitative determination of the bacteria, fungiand yeasts. In a preferred embodiment, this analysis is performed in onestep.

Following the separation of microorganisms and minerals, fillers,pigments and/or fiber materials, i.e. the “foreign particles”, one ormore, preferably two to five incubation steps are carried out toincrease the number of microorganisms present in the sample supernatant.

Multiple incubations are performed in intervals, i.e. the measurement ofthe contamination is in each case repeated after the time t_(0+x). Thisenables the limitation of the incubation time and the incubationintervals. At the time when exponential growth is observed anextrapolation and a calculation back to the time t₀ is performed. Thus,a high original degree of contamination will result in a growth whichcan be observed and calculated earlier so that subsequent incubationintervals may be omitted.

The incubation time at a contamination of less than 10⁵ germs/ml ofsample is up to 12 hours, at a contamination of less than 10⁶ germs/mlup to 6 hours, in a suspension originally containing 60-80% by wt. ofsolids and having a contamination of more than 10⁵ germs/ml sample up to3 hours wherein in the last case the use of tryptic soy broth agar ispreferred. The incubation time which is to be used depending on thedegree of contamination with microorganisms may be determined by thoseskilled in the art by simple manual experimentation. The lower theoriginal degree of microbial contamination in the sample the longer arethe incubation times to be selected and vice versa. The original portionof solids in suspension does not directly affect the incubation time.

In the following, the invention will be explained in more detail withrespect to comparative examples and Examples. Further modifications ofthe invention may be practiced by the skilled artisan on the basis ofthe present Specification in connection with the appended Claims as wellas on the basis of assumed expertise. The invention is not limited tothe present examples.

Determination of the Germ Count Using the CellFacts® Particle Counter

According to the present invention, the microorganisms of the suspensionand/or emulsion and/or dispersion are aspirated by means of vacuumthrough a measurement pore having a defined diameter. A voltage (Volts)is applied to this pore (capillary, 30 μm). Because intact cells in afirst approximation can be regarded as insulators a measurable increasein resistance is observed during passage of a cell through themeasurement pore while the value of this increase is dependent on thecell volume. The current pulse is directly correlated to the volume. Byincubation of the samples, small differences in the growth of the cellscan be measured, i.e. cell divisions lead to a proportional increase inparticle number. After observation of the exponential phase, thestarting concentration may be calculated by extrapolation because thecell growth function is exponential. Due to the preparation ofindustrial samples according to the present invention and using theabove method the determination time for CaCO₃, talc and kaolin slurriesas well as other materials (e.g. coolants, white water, colloidalsuspended starch, etc.) is in the range of only 2-12 hours instead of24-48 hours of the prior art.

This enables timely intervention using preservatives prior to highcontamination and/or spoilage of the goods.

PRIOR ART EXAMPLES Example 1 Calcium Carbonate Slurry

A) Germ count determinations according to the SchweizerischesLebensmittelbuch, chapter 56, section 7.01, 1985 edition, 1988 revisedversion, “Bestimmung von aeroben Bakterien und Keimen” and according tothe Schweizerisches Lebensmittelbuch, chapter 56, section 7.22, 1985edition, 1988 revised version, “Bestimmung von Pilzen”.

Proceeding

3 ml of a 77.5% by wt. aqueous slurry of natural ground marble fromNorway (90% by wt. of the particles <2 μm, 65% by wt. of the particles<1 μm) dispersed with 0.65% by wt. of a commercial sodium polyacrylatewere analyzed according to the method “Bestimmung von aeroben mesophilenKeimen”, Schweizerisches Lebensmittelbuch, chapter 56, section 7.01,1985 edition, 1988 revised version.

Result:

After an incubation period of 48 hours the germ count determined was:3.0×10⁶ aerobic mesophilic germs/ml of suspension. No yeasts were foundin the nutrient medium used for aerobic mesophilic germs. According tothe method described in the Schweizerisches Lebensmittelbuch, chapter56, section 7.22, 1985 edition, 1988 revised version, “Bestimmung vonPilzen”, <10² fungi/ml were detected.

B) Determination of the Germ Count Using the CellFacts Particle Counter

Proceeding

3 ml of the 77.5% by wt. aqueous slurry of natural ground marble fromNorway (90% by wt. of the particles <2 μm, 65% by wt. of the particles<1 μm) dispersed with 0.65% by wt. of a commercial sodium polyacrylateused in Example 1A) were pipetted into a sterile flask. The sample wasadded with 3 ml of a 3% by wt. TAT broth base/Tween 20 nutrient solutionand analyzed by means of CellFacts. The analysis was performed byimmediately attempting the measurement of the number of particles in theCellFacts instrument (time t_(0h)). Since the number of particles wasmuch too high the instrument required a sample dilution. After adilution of 1:10,000 had been carried out, the particle concentrationwas accepted by the instrument, and the blank value (t_(0h)) could bedetermined. After incubation times of 2 hrs. (time t_(2h)), 4 hrs. (timet_(4h)), after 6 hrs. (time t_(6h)), after 12 hrs. (time t_(12h)) thenumber of particles was again determined in the CellFacts instrument.The incubation temperature was 30° C. By plotting the number ofparticles against the particle diameter it is possible to determine thepresence of living cells (“peak enlargement”, y axis) and the type ofthe microorganisms, e.g. bacteria, yeasts or fungi (mean particle ø, xaxis) after particular incubation times of the clear phase.

After an incubation time of 12 hours the original contamination of thesuspension is evaluated by extrapolating back to the time (t_(0h)). As areference, the result of Example 1A), germ count determination accordingto Schweizerisches Lebensmittelbuch, is used. (“Bestimmung von aerobenmesophilen Keimen”, Schweizerisches Lebensmittelbuch, chapter 56,section 7.01, 1985 edition, 1988 revised version.

Result:

Germ Count Determination Using the CellFacts Particle Counter

1. Tryptic Soy Broth Agar

Aerobic mesophilic germs/ml of suspension (peak Extraction Determinationmax. at particle Ø Sample agent time in hrs. of 1-2.5 μm) 1 Tryptic soy12 <10² broth agar fungi/ml of suspension (peak Extraction Determinationmax. at particle Ø Sample agent time in hrs. of 5-9 μm) 1 Tryptic soy 12<10² broth agar

It becomes clear from Examples 1A) and 1B) that using the methods of theprior art it is impossible to perform a determination of bacteria, fungiand yeasts in the above suspension by means of the CellFacts instrument,and that particularly at <10² germs/ml a completely inaccurate result isobtained. Furthermore, the determination according to the method of theSchweizerisches Lebensmittelbuch requires 48 hrs.

Presumably, by the sample dilution required in the CellFacts instrumentalso the microbiological germs present were diluted to below thedetection limit.

Performing the determination according to the SchweizerischesLebensmittelbuch the duration of the analysis is 48 hrs.

C) Germ Count Determination by ATP Measurement

Proceeding

3 ml of the sample of Example 1A), 77.5% by wt. aqueous slurry ofnatural ground marble from Norway (90% by wt. of the particles <2 μm,65% by wt. of the particles <1 μm) dispersed with 0.65% by wt. of acommercial sodium polyacrylate, were pipetted into a sterile flask. Thesample was added with 3 ml of a 3% by wt. TAT broth base/Tween 20nutrient solution and analyzed by means of the BioOrbit lunimometer1253. The analysis was performed by immediately attempting themeasurement of the amount of light released by ATP in the BioOrbitluminometer 1253 (time t_(0h)). In addition, a sample was measured afterperforming a 1:10,000 dilution. In this manner, the blank value (t_(0h))was determined in each case. After incubation times of 4 hrs. (timet_(4h)), after 7 hrs. (time t_(7h)), and after 17 hrs. (time t_(17h))measurements of the light intensity in the BioOrbit 1253 luminometerwere carried out. The incubation temperature was 30° C. By plotting thelight intensity at the respective incubation time against the incubationtime it is possible to determine the presence of living cells (increasein light intensity over time). After an incubation time with nearlyexponential growth the contamination of the suspension may be determinedcomparatively by comparing the progression of the curves of theindividual samples. By this, a semi-quantitative distinction may be madebetween “no growth”, “weak growth”, and “strong growth”.

Proceeding of the Measurement in the Luminometer 1253

a) Initially, 100 μl of each of the samples prepared as described aboveare pipetted into a luminometer cuvette.

b) To this, 100 μl of ATP releasing reagent are added, shook well andleft for one minute so that the ATP may be removed by extraction.

c) Afterwards, 500 μl of AMR reagent are added and mixed by shaking.

d) Then, the cuvette may be introduced into the measurement cell andreadings of the light emission may be performed.

Reference: User's manual of the BioOrbit luminometer ver. 3.0, May 95 aswell as Application Note 20 of BioOrbit OY company, FIN 20521 Turku,Finland.

Result:

Sample 1:10,000 Sample light dilution, light Incubation time intensity[RLU] on intensity [RLU] on in hours the LCD display the LCD display 00.001 0.002 4 0.000 0.001 7 0.002 0.002 17  0.001 0.001

After 17 hours, no differences between the individual incubation timescan be observed using this prior art method, i.e. it has to be assumedthat the slurry is not contaminated. However, the same slurry has beenused in Example 1A) (method according to SchweizerischesLebensmittelbuch), and since this appreciated method used in the Examplegives a result of 3×10⁶ germs/ml it becomes obvious that the result ofthe present Example 1C) must be erroneous. In practice, this wouldentail fatal consequences such as food poisoning or spoilage of goods.

Using the methods of the prior art, it is impossible to determinebacteria, fungi and yeasts in the above-mentioned suspension.

Presumably, the high particle concentration in the originalconcentration of the suspension resulted in complete light adsorption ofthe amount of light released by ATP in the suspension, and sampledilution by this method also led to a dilution of the microbiologicalgerms present to below the detection limit thereof.

INVENTIVE EXAMPLES Example 2 Calcium Carbonate Slurry, AnalysisInstrument CellFacts Measuring Instrument

Proceeding

3 ml each of the 77.5% by wt. aqueous slurry of natural ground marblefrom Norway (90% by wt. of the particles <2 μm, 65% by wt. of theparticles <1 μm) dispersed with 0.65% by wt. of a commercial sodiumpolyacrylate are pipetted into separate sterile flasks. One sample isadded with 3 ml of a 3% by wt. TAT broth base/Tween 20 nutrient solutionand the second sample with 3 ml of a 3% by wt. tryptic soy brothnutrient solution. Afterwards, both of the samples are mixed well for 20sec. and then centrifuged. After a centrifugation for 10 min at 800 gthe clear supernatant phase is isolated and analyzed by means ofCellFacts. The analysis is performed by measuring the number ofparticles in the CellFacts instrument immediately after centrifugation(time t_(0h)), after incubation times of 2 hrs. (time t_(2h)), 4 hrs.(time t_(4h)), and after 6 hrs. (time t_(6h)). The incubationtemperature was 30° C. By plotting the number of particles against theparticle diameter it is possible to determine the presence of livingcells (peak enlargement) and the type of the microorganisms (bacteria,yeasts or fungi) after particular incubation times of the clear phase.After an incubation time with exponential growth the originalcontamination of the suspension is determined by extrapolating back tothe time t_(0h). In comparison, the germ count determination accordingto the Schweizerisches Lebensmittelbuch is performed. (“Bestimmung vonaeroben mesophilen Keimen”, Schweizerisches Lebensmittelbuch, chapter56, section 7.01, 1985 edition. 1988 revised version).

Results:

Germ Count Determination According to the SchweizerischesLebensmittelbuch

The germ count determination after 48 hours revealed: 3.0×10⁶ aerobicmesophilic germs/ml of suspension

Germ Count Determination Using the CellFacts Particle Counter

1. TAT broth base/Tween 20 (polyoxyethylene sorbitan monolaurate)

2. Tryptic soy broth agar

Aerobic mesophilic germs/ml of Required suspension (peak ExtractionDetermination max. at particle Ø Sample agent time in hrs. of 1-2.5 μm)1 TAT broth 6 1.25 × 10⁶ base/ Tween 20 2 Tryptic soy 4 3.14 × 10⁶ brothagar fungi/ml of suspension (peak Extraction Determination max. atparticle Ø Sample agent time in hrs. of 5-9 μm) 2 Tryptic soy 12 <10²broth agar

The samples analyzed according to the method of the present inventionare in excellent accordance with the determination of the germ countaccording to the Schweizerisches Lebensmittelbuch determined after 48hours. Compared to the determination of the germ count according to theSchweizerisches Lebensmittelbuch both samples are very well within theconfidence limits.

Confidence limits, as used herein, is intended to mean a deviation of±0.5 log₁₀ of individual germ count determinations as established by thePHLS Central Public Health Laboratory, 61 Colindale Avenue, London NW95HT, UK, distribution 070, pages 6 and 7 and appendix 1, distributiondate Nov. 16, 1998, report date Dec. 30, 1998.

Example 3 Calcium Carbonate Slurry, Analysis Device BioOrbit 1253Luminometer

Proceeding

500 ml each of a 71.5% by wt. aqueous slurry of natural ground marble(90% by wt. of the particles <2 μm, 65% by wt. of the particles <1 μm)dispersed with 0.5% by wt. of a commercial sodium polyacrylate, werecharged into 1 l glass flasks. One of the samples was stored for 3 daysat 5° C. (sample A), the other was stored for 3 days at 30° C. (sampleB). Afterwards, the samples were brought to 20° C., and 3 ml of each ofthe samples were mixed well with 3 ml of the respective extraction agentin sterile flasks. Following a centrifugation for 10 min at 600 g theclear supernatant phase is isolated and analyzed for light emission bymeans of the BioOrbit lunimometer 1253. The analysis is performed bymeasuring the light intensity in the BioOrbit luminometer 1253immediately after centrifugation (time t_(0h)), after 4 hrs. (timet_(4h)), after 7 hrs. (time t_(7h)), after 17 hrs. (time t_(17h)). Theincubation temperature was 30° C. By plotting the light intensity afterthe respective incubation time against the incubation time it ispossible to determine the presence of living cells (increase in lightintensity over time) in the clear phase. After an incubation time withnearly exponential growth the contamination of the suspension may bedetermined comparatively by comparing the progression of the curves ofthe individual samples. By this, a semi-quantitative distinction may bemade between “no growth”, “weak growth”, and “strong growth”. Incomparison, a germ count determination according to the SchweizerischesLebensmittelbuch was performed. (“Bestimmung von aeroben mesophilenKeimen”, Schweizerisches Lebensmittelbuch, chapter 56, section 7.01,1985 edition. 1988 revised version).

Extraction agents used:

1. Prior art, 0.9% by wt. solution of NaCl in Aq. dest. without organicsubstance that may serve as a nutrient

2. According to the invention, 3% by wt. organic nutrient solution(tryptic soy broth agar)

Germ Count Determination According to the SchweizerischesLebensmittelbuch

Sample A: 5×10⁴ aerobic mesophilic germs/g of suspension after 48 hours

Sample B: 7×10⁷ aerobic mesophilic germs/g of suspension after 48 hours

Results:

Evaluation of the contamination with the BioOrbit luminometer

Prior art, 0.9% by wt. solution of NaCl in Aq. dest.

Sample 1A, Sample 1B, light light Incubation time intensity [RLU] onintensity [RLU] on in hours the LCD display the LCD display 0 0.0010.001 4 0.002 0.003 7 0.002 0.004 17  0.004 0.007

Using the prior art method, no clear difference is observed between theweakly and the strongly contaminated suspension after 17 hours.

According to the present invention, 3% by wt. organic nutrient solution(tryptic soy broth agar)

Sample 1A, light Sample 1B, light Incubation time intensity [RLU] onintensity [RLU] on in hours the LCD display the LCD display 0 0.0010.002 4 0.004 0.006 7 0.007 0.045 17  0.050 0.240

Using the novel method according to the present invention, it ispossible to distinguish between “weakly” and “strongly contaminated”solutions already after 7 hours. In addition, a “weak contamination” maybe significantly detected already after 17 hours.

The literature and the user's manual of the BioOrbit luminometer ver.3.0, May 95 as well as “application note 20” of BioOrbit OY company, FIN20521 Turku, Finland, also show evaluation models for the calculation ofthe biomass using the [RLU] values. Furthermore, the germ count/ml maybe determined by “calibration” using suspensions with a known degree ofcontamination.

Example 4 US Clay Slurry

Proceeding

3 ml each of a 72.8% by wt. aqueous slurry of natural ground US clayfrom Georgia USA (95% by wt. of the particles <2 μm, 78% by wt. of theparticles <1 μm) dispersed with 0.35% by wt. of a commercial sodiumpolyacrylate are pipetted into separate sterile flasks. One sample isadded with 3 ml of a 3% by wt. TAT broth base/Tween 20 nutrient solutionand the second sample with 3 ml of a 3% by wt. tryptic soy brothnutrient solution. Afterwards, both of the samples are mixed well for 20sec. and then centrifuged. After a centrifugation for 10 min at 800 gthe clear supernatant phase is isolated and analyzed by means ofCellFacts. The analysis is performed by measuring the number ofparticles in the CellFacts instrument immediately followingcentrifugation (time t_(0h)), after incubation times of 2 hrs. (timet_(2h)), 4 hrs. (time t_(4h)), after 6 hrs. (time t_(6h)), and after 12hrs. (time t_(12h)). The incubation temperature was 30° C. By plottingthe number of particles against the diameter of these particles it ispossible to determine the presence of living cells (peak enlargement)and the species of the microorganisms (bacteria, yeasts or fungi) afterparticular incubation times of the clear phase. After an incubation timewith nearly exponential growth the original contamination of thesuspension is determined by extrapolating back to the time t_(0h). Incomparison, a germ count determination according to the SchweizerischesLebensmittelbuch is performed. (“Bestimmung von aeroben mesophilenKeimen”, Schweizerisches Lebensmittelbuch, chapter 56, section 7.01,1985 edition. 1988 revised version).

Germ Count Determination According to the SchweizerischesLebensmittelbuch

The germ count determination after 48 hours revealed: 5.0×10⁵ aerobicmesophilic germs/ml of suspension

Germ Count Determination Using the CellFacts Particle Counter

1. TAT broth base/Tween 20 (polyoxyethylene sorbitan monolaurate)

2. Tryptic soy broth agar

Aerobic mesophilic germs/ml of Required suspension (peak ExtractionDetermination max. at particle Ø Sample agent time in hrs. of 1-2.5 μm)1 TAT broth 12 1.12 × 10⁵ base/ Tween 20 2 Tryptic soy  6 5.21 × 10⁵broth agar fungi/ml of suspension (peak Extraction Determination max. atparticle Ø Sample agent time in hrs. of 5-9 μm) 2 Tryptic soy 12 approx.10³ broth agar

The samples analyzed according to the method of the present inventionare in excellent accordance with the determination of the germ countaccording to the Schweizerisches Lebensmittelbuch determined after 48hours. Compared to the determination of the germ count according to theSchweizerisches Lebensmittelbuch both samples are very well within theconfidence limits.

Confidence limits, as used herein, is intended to mean a deviation of±0.5 log₁₀ of individual germ count determinations as established by thePHLS Central Public Health Laboratory, 61 Colindale Avenue, London NW95HT, UK, distribution 070, pages 6 and 7 and appendix 1, distributiondate Nov. 16, 1998, report date Dec. 30, 1998.

Example 5 Paper Making Machine White Water

Proceeding

3 ml of a 0.5% by wt. aqueous slurry containing about 0.2% by wt. ofnatural ground marble from Norway (60% by wt. of the particles <2 μm,35% by wt. of the particles <1 μm) dispersed with 0.15% by wt. of acommercial sodium polyacrylate and about 0.3% by wt. of cellulose fibers(sulfite/sulfate paper pulp) as it is used in paper mills for papermaking and about 0.05% by wt. of a commercial polyacrylamide arepipetted into a sterile flask. The sample is added with 3 ml of a 3% bywt. tryptic soy broth nutrient solution. Afterwards, the sample is mixedwell for 20 sec. and then centrifuged. After a centrifugation for 10 minat 1000 g the clear supernatant phase is isolated and analyzed by meansof CellFacts. The analysis is performed by measuring the number ofparticles in the CellFacts instrument immediately followingcentrifugation (time t_(0h)), after incubation times of 1 hr. (timet_(1h)), 2 hrs. (time t_(2h)). The incubation temperature was 30° C. Byplotting the number of particles against the diameter of these particlesit is possible to determine the presence of living cells (peakenlargement) and the species of the microorganisms (bacteria, yeasts orfungi) after particular incubation times of the clear phase. After anincubation time with nearly exponential growth the originalcontamination of the suspension is determined by extrapolating back tothe time t_(0h). In comparison, a germ count determination according tothe Schweizerisches Lebensmittelbuch is performed. (“Bestimmung vonaeroben mesophilen Keimen”, Schweizerisches Lebensmittelbuch, chapter56, section 7.01, 1985 edition. 1988 revised version).

Germ Count Determination According to the SchweizerischesLebensmittelbuch

The germ count determination after 48 hours revealed: 3.0×10⁷ aerobicmesophilic germs/ml of suspension

Germ Count Determination Using the CellFacts Particle Counter

1. Tryptic soy broth agar

Aerobic mesophilic germs/ml of Required suspension (peak ExtractionDetermination max. at particle Ø Sample agent time in hrs. of 1-2.5 μm)1 Tryptic soy 2 3.45 × 10⁷ broth agar fungi/ml of suspension (peakExtraction Determination max. at particle Ø Sample agent time in hrs. of5-9 μm) 1 Tryptic soy 6 approx. 10⁴ broth agar

The samples analyzed according to the method of the present inventionare in excellent accordance with the determination of the germ countaccording to the Schweizerisches Lebensmittelbuch determined after 48hours. Compared to the determination of the germ count according to theSchweizerisches Lebensmittelbuch both samples are very well within theconfidence limits.

Confidence limits, as used herein, is intended to mean a deviation of±0.5 log₁₀ of individual germ count determinations as established by thePHLS Central Public Health Laboratory, 61 Colindale Avenue, London NW95HT, UK, distribution 070, pages 6 and 7 and appendix 1, distributiondate Nov. 16, 1998, report date Dec. 30, 1998.

Example 6 Colloidal Starch Suspension

Proceeding

3 ml of a 10% by wt. aqueous colloidal corn starch suspension asemployed in paper mills are pipetted into a sterile flask. The sample isadded with 3 ml of a 3% by wt. tryptic soy broth nutrient solution.Afterwards, the sample is mixed well for 20 sec. and then centrifuged.After a centrifugation for 10 min at 800 g the clear supernatant phaseis isolated and analyzed by means of CellFacts. The analysis isperformed by measuring the number of particles in the CellFactsinstrument immediately following centrifugation (time t_(0h)), afterincubation times of 1 hr. (time t_(1h)), 2 hrs. (time t_(2h)). Theincubation temperature was 30° C. By plotting the number of particlesagainst the diameter of these particles it is possible to determine thepresence of living cells (peak enlargement) and the species of themicroorganisms (bacteria, yeasts or fungi) after particular incubationtimes of the clear phase. After an incubation time with nearlyexponential growth the original contamination of the suspension isdetermined by extrapolating back to the time t_(0h). In comparison, agerm count determination according to the SchweizerischesLebensmittelbuch is performed. (“Bestimmung von aeroben mesophilenKeimen”, Schweizerisches Lebensmittelbuch, chapter 56, section 7.01,1985 edition. 1988 revised version).

Germ Count Determination According to the SchweizerischesLebensmittelbuch

The germ count determination after 48 hours revealed: 4.0×10⁶ aerobicmesophilic germs/ml of suspension

Germ Count Determination Using the CellFacts Particle Counter

1. Tryptic soy broth agar

Aerobic mesophilic germs/ml of Required suspension (peak ExtractionDetermination max. at particle Ø Sample agent time in hrs. of 1-2.5 μm)1 Tryptic soy  2 4.22 × 10⁶ broth agar fungi/ml of suspension (peakExtraction Determination max. at particle Ø Sample agent time in hrs. of5-9 μm) 1 Tryptic soy 12 <10² broth agar

The sample analyzed according to the method of the present invention arein excellent accordance with the determination of the germ countaccording to the Schweizerisches Lebensmittelbuch determined after 48hours. Compared to the determination of the germ count according to theSchweizerisches Lebensmittelbuch both samples are very well within theconfidence limits.

Confidence limits, as used herein, is intended to mean a deviation of±0.5 log₁₀ of individual germ count determinations as established by thePHLS Central Public Health Laboratory, 61 Colindale Avenue, London NW95HT, UK, distribution 070, pages 6 and 7 and appendix 1, distributiondate Nov. 16, 1998, report date Dec. 30, 1998.

Example 7

Proceeding

3 ml each of a 71.5% by wt. aqueous slurry of natural ground marble (90%by wt. of the particles <2 μm, 65% by wt. of the particles <1 μm)dispersed with 0.5% by wt. of a commercial sodium polyacrylate are mixedwell in sterile flasks with 3 ml each of the respective extractionagent. After a centrifugation for 10 min at 800 g the clear supernatantphase is isolated and analyzed by means of CellFacts. The analysis isperformed by measuring the number of particles in the CellFactsinstrument immediately following centrifugation (time t_(0h)), afterincubation times of 2 hrs. (time t_(2h)), 4 hrs. (time t_(4h)), after 7hrs. (time t_(7h)), after 17 hrs. (time t_(17h)), and after 24 hrs.(time t_(24h)). The incubation temperature was 30° C. By plotting thenumber of particles against the diameter of these particles it ispossible to determine the presence of living cells (peak enlargement)and the species of the microorganisms (bacteria, yeasts or fungi) afterparticular incubation times of the clear phase. After an incubation timewith nearly exponential growth the original contamination of thesuspension is determined by extrapolating back to the time t_(0h). Incomparison, a germ count determination according to the SchweizerischesLebensmittelbuch is performed. (“Bestimmung von aeroben mesophilenKeimen”, Schweizerisches Lebensmittelbuch, chapter 56, section 7.01,1985 edition. 1988 revised version).

Extraction agents used:

Prior Art

1. Aq. dest.

2. 0.9% by wt. NaCl solution in Aq. dest., inorganic salt

3. 0.8% by wt. solution of polyoxyethylene sorbitan monolaurate (2 molesof polyoxyethylene), organic surfactant

Example According to the Present Invention

4. 3% by wt. of organic nutrient solution (tryptic soy broth agar)

Germ Count Determination According to the SchweizerischesLebensmittelbuch

The germ count determination after 48 hours revealed: 5.0×10⁴ aerobicmesophilic germs/ml of suspension

Germ Count Determination Using the CellFacts Particle Counter

Aerobic mesophilic germs/ml of Required suspension (peak ExtractionDetermination max. at particle Ø Sample agent time in hrs. of 1-2.5 μm)1 Aq. dest. 17   2 × 10³ 2 NaCl solution 18 1.6 × 10³ 3 polyoxyethylene24 >10² sorbitan monolaurate 4 organic nutrient  7 5,6 × 10⁴ solution

The samples 1-3 first clearly demonstrate that a very long incubationtime is required until a result can be calculated and, second, that alsothis result is incorrect. (Reference: determination of the germ countaccording to the Schweizerisches Lebensmittelbuch)

For samples 1-3, the variation to the germ count determined according tothe Schweizerisches Lebensmittelbuch is far beyond the confidence limit.

Samples 1 and 2 lack an organic substance which may act as a nutrient.Sample 3 contains an organic substance. However, this substance is notactive as a nutrient but rather as an inhibitor. A certain inhibition byTween 20 can already be observed in Example 4-1 where Tween 20 has beenemployed if the result is compared to Example 4-2. However, the organicsubstance acting as a nutrient solution employed according to thepresent invention was able to prevent an incorrect result in the case ofExample 4-2. However, in comparison to Example 7-4, the Example 7-3gives fatal erroneous result!

Sample 4 which has been analyzed according to the method of the presentinvention is in excellent accordance with the germ count determinationaccording to the Schweizerisches Lebensmittelbuch.

In sample 4, the difference to the germ count determination according tothe Schweizerisches Lebensmittelbuch is very well within the confidencelimits.

Confidence limits, as used herein, is intended to mean a deviation of±0.5 log₁₀ of individual germ count determinations as established by thePHLS Central Public Health Laboratory, 61 Colindale Avenue, London NW95HT, UK, distribution 070, pages 6 and 7 and appendix 1, distributiondate Nov. 16, 1998, report date Dec. 30, 1998.

Composition of Preferred Nutrient Media

Tryptic Soy Broth Bacto tryptone 17.0 g/l Bacto soytone 3.0 g/l Bactodextrose 2.5 g/l sodium chloride 5.0 g/l dipotassium phosphate 2.5 g/l

TAT Broth Base Bacto tryptone 20.0 g/l azolectin 5.0 g/l

Nutrient Broth Bacto beef extract 3.0 g/l Bacto peptin 5.0 g/l

Peptin from casein, tryptic digest peptin from casein 1.0 g/l sodiumchloride 1.8 g/l

Plate Count Agar Bacto tryptone 5.0 g/l yeast extract 2.5 g/l Bactodextrose 1.0 g/l Agar No. 1 9.0 g/l

Ethylene Glycol ethylene glycol 1.0 g/l azolectin 5.0 g/l sodiumchloride 1.8 g/l

Glycerol glycerol 1.0 g/l azolectin 5.0 g/l sodium chloride 1.8 g/l

What is claimed is:
 1. A method for the quantitative or qualitativedetermination of microbial contamination of a suspension, emulsion, ordispersion comprising a mineral, pigment, filler, fiber material, orcombinations thereof, the method comprising: (a) mixing a sample of thesuspension, emulsion, or dispersion with an amount of one or moreorganic substances which can be degraded by a microorganism and which iseffective as a separating agent between the microorganism and themineral, pigment, filler, fiber material, or combinations thereof,wherein the amount of one or more organic substances is selected suchthat a separation of the microorganism from the mineral, filler,pigment, fiber material, or combinations thereof is rendered possible;(b) centrifuging the mixture obtained in step (a) so that a majority ofthe mineral, filler, pigment, fiber material, or combinations thereof isseparated from the microorganism and the microorganism is in the upperphase; (c) separating the upper phase as an aqueous supernatant; and (d)determining the number, size, type, or combinations thereof, of themicroorganism in the supernatant.
 2. The method according to claim 1,further comprising performing an incubation in step (a) to increase thenumber of the microorganism.
 3. The method of claim 2, wherein theincubation temperature is 20-37° C.
 4. The method of claim 3, whereinthe incubation temperature is 28-34° C.
 5. The method of claim 4,wherein the incubation temperature is 31.5-32.5° C.
 6. The method ofclaim 1, wherein the microbial contamination is selected from the groupconsisting of bacteria, fungi, yeasts, and combinations thereof.
 7. Themethod of claim 6, wherein the determination of bacteria, fungi, yeasts,and combinations thereof is qualitative or quantitative and is performedsimultaneously in one analysis.
 8. The method of claim 1, furthercomprising subjecting the aqueous supernatant of step (c) to one or moreincubations in order to increase the number of the microorganism in thesupernatant.
 9. The method of claim 1, wherein the one or more organicsubstances is added in the form of a nutrient medium for microorganisms.10. The method of claim 9, wherein the nutrient medium comprises asource of carbon, nitrogen, phosphate, sulphur, or combinations thereof.11. The method of claim 9, wherein the nutrient medium further comprisesa mineral, a growth factor, a vitamin, or combinations thereof.
 12. Themethod of claim 1, wherein the centrifugation is performed at 100-1,500g.
 13. The method of claim 12, wherein the centrifugation is performedat 200-1,200 g.
 14. The method of claim 13, wherein the centrifugationis performed at 600-1,000 g.
 15. The method of claim 1, furthercomprising using a nutrient medium that enables the selective growth ofthe microorganism to be determined.
 16. The method of claim 15, whereinthe nutrient medium is selected from the group consisting of tryptoneazolectin/Tween 20 broth agar, glucose solution, peptone/casein,nutrient broth, and combinations thereof.
 17. The method of claim 16,wherein the nutrient broth is tryptic soy broth agar.
 18. The method ofclaim 1, wherein the concentration of the nutrient medium is 0.1-10% byweight.
 19. The method of claim 18, wherein the concentration of thenutrient medium is 2-5% by weight.
 20. The method of claim 19, whereinthe concentration of the nutrient medium is 3% by weight.
 21. The methodof claim 1, wherein the suspension, emulsion, or dispersion furthercomprises a polymer in a colloidal form.
 22. The method of claim 1,wherein the centrifugation step is performed for a time of 1-30 minutes.23. The method of claim 22, wherein the centrifugation step is performedfor a time of 2-15 minutes.
 24. The method of claim 23, wherein thecentrifugation step is performed for a time of 10 minutes.
 25. Themethod of claim 1, further comprising analyzing the upper phase obtainedby centrifugation containing the microorganism for the number of themicroorganism by a particle size analysis method.
 26. The method ofclaim 25, wherein the size and number of the microorganism are analyzedby aspirating the microorganism sampled using a vacuum through ameasurement pore having a defined length and diameter, wherein a voltageis applied at the pore inlet and exit and a current pulse is measuredupon passage of the microorganism through the sample which is directlycorrelated to the volume of the microorganism, and the number of pulsesis correlated to the number of the microorganism.
 27. The method ofclaim 1, further comprising analyzing the upper phase obtained bycentrifugation containing the microorganism for the number ofmicroorganisms by determining an amount of ATP produced by themicroorganisms.
 28. A method of investigating the microbialcontamination of a dispersion, emulsion, or suspension in a metalindustry, pigment and paper industry, or paper industry white waters,the method comprising: (a) mixing a sample of the suspension, emulsion,or dispersion with an amount of one or more organic substances which canbe degraded by a microorganism and which is effective as a separatingagent between the microorganism and a mineral, pigment, filler, fibermaterial, or combinations thereof, wherein the amount of the one or moreorganic substances is selected in a manner that a separation of themicroorganism from the mineral, filler, pigment, fiber material, orcombinations thereof is rendered possible; (b) subjecting the mixturethus obtained to centrifugation so that a majority of the mineral,filler, pigment, fiber material, or combinations thereof is separatedfrom the microorganism and the microorganism is in the upper phase; (c)separating the upper phase as an aqueous supernatant; and (d)determining the number, size, type, or combinations thereof, of themicroorganism in the supernatant.